The easternmost Tian Shan lies in eastern Xinjiang, Central Asia. The South Barkol basin fault (SBF) in the northern part of the easternmost Tian Shan is a major tectonic structure in this orogenic region. The late Quaternary activity, paleoseismology, and deformation characteristics of the fault provide important clues for understanding the tectonic process of the eastern Tian Shan orogen and implementing seismic mitigation. Through interpretation of high-resolution satellite images, unmanned aerial vehicle measurements, and detailed geological and geomorphic investigations, we suggest that the fault exhibits clear left-lateral slip along its western segment. Paleoseismic trenches dug near Xiongkuer reveal evidence of six large paleoearthquakes. The four latest paleoearthquakes were dated: the oldest event occurred at 4663 BC–3839 BC. Data on the horizontal offsets along the probable 1842 Barkol earthquake coseismic rupture suggest clear multiple relationships between cumulative offsets and possible ~4 m of coseismic left-lateral slip per event. From the cumulative offsets and 14C sample ages, we suggest an average Holocene left-lateral slip rate of 2.4–2.8 mm/a on the SBF, accounting for ~80% of lateral deformation within the entire eastern Tian Shan fault system. This result is comparable with the shortening rate of 2–4 mm/a in the whole eastern Tian Shan, indicating an equal role of strike-slip tectonics and compressional tectonics in this orogen, and that the SBF may accommodate substantial lateral tectonic deformation.